plasmid profile analysis of salmonella enterica serotype enteritidis

Review article
PLASMID PROFILE ANALYSIS OF SALMONELLA ENTERICA SEROTYPE
ENTERITIDIS
54
Biljana Miljkovic-Selimovic, Tatjana Babic, Branislava Kocic, Predrag Stojanovic, Ljiljana Ristic and
Marina Dinic
Plasmid profile analysis (PP) is a method of determining a number and size of
plasmids in bacterial isolates. When using plasmid profile analysis in determining
epidemiological strain for evaluation of this method, it is necessary to compare
epidemiological strains with non-epidemiological ones isolated in the same period of
time. Plasmid profile determination is performed by lysis of bacterial cell,
chromosomal denaturation, sedimentation of cell fragments by centrifugation, and
precipitation of DNA with ethanol. DNA is separated by gel electrophoresis based on its
molecule mass. Plasmids could be also analyzed by restriction enzymes, as well as by
direct techniques, such as hybridization and electronomicroscopically. Using of plasmid
profile analysis enables resolving numerous outbreaks caused by Enterobacteriaceae,
especially Salmonella. This method could be performed alone for Salmonella enterica
serotype Enteritidis, but also as a complementary method to phage typing
(phagotypization), and other molecular genetic based techniques. Acta Medica
Medianae 2008;47(2):54-57.
Key words: plasmid profile, Salmonella enterica serotype Enteritidis
Public Health Institute Clinical Center in Nis
Contact: Biljana Miljkovic-Selimovic
Public Health Institute Clinical Center
50 Dr Zoran Djindjic Blvd.
18000 Nis, Serbia
Phone:+381 18 226448; 0644332238
E-mail: biljams@eunet.yu
Introduction
Since ‘70s of the past century, along with
improving methods of plasmids isolation, the
importance of researching techniques of total
bacterial plasmids contents as a mean of determining
clone concept rose up. Determination of a
number and size of plasmids in bacterial isolates
is called plasmid profile analysis (PP) (1,2,3,4). It
appears that epidemical strains of Enterobacteriaceae,
bacteria of Pseudomonas and Vibrio
strains, staphylococcus and streptococcus can be
identified in this way (4,5). However, isolates
which are not connected with the outbreak may
manifest epidemical profile. Thus, when using
plasmid profile analysis in determining epidemical
strain for evaluation of this method, it is
necessary to compare epidemiological strains
with non-epidemiological ones isolated in the
same period of time (4).
Namely, determination of plasmids profile
is performed after the growth of culture in a
liquid or on a solid medium, by breaking off outer
membrane of bacterial cell with lysosomes, inner
membrane by detergent lysis, and chromosomes
denaturation in alkali pH. Cell debris is sedimented
by centrifugation in a microcentrifuge for a
several minutes, after which DNA precipitation
with ethanol follows. This method, modified to
some degree, can be used for isolation of plasmid
DNA from majority of genera and strains related
to family of Enterobacteriaceae (4). Plasmid DNA
is exposed to horizontal or vertical gel electrophoresis,
while process of separation, based on
molecule mass, occurs during migration toward
anode. Additionally, gel is painted with ethidium
bromide which binds to DNA and shows the
fluorescent color when exposed to UV light.
Plasmid DNA with known molecule mass is
recognized as a standard for every gel and its
relative mobility is used in calculation of mass of
unknown plasmids (4). As the length of migration
relies on shape and dimension of plasmids, we
can find more strips of plasmids on gel. Coiled
molecules with covalent connections, which
migrate at the highest speed, during extraction
could be changed to open ring-shaped (circular)
or linear forms. Since the coiled forms migrate
faster than relaxed, their mutual relation and
position in electrophoresis field are influenced by
electric power, puffer system, the sizes of gel
pores and DNA molecules. Therefore, appearing
of several stripes could be interpreted in wrong
manner as the existence of large number of
www.medfak.ni.ac.yu/amm

Acta Medica Medianae 2008,Vol.47 Plasmid profile analysis of salmonella enterica serotype enteritidis
plasmids, instead of multiple forms of one single
plasmid. On the other hand, as the electrophoresis
mobility of two different plasmids could be
the same, it is necessary to determine whether,
based on examination of sequences of nucleotide
bases, these plasmids have the same molecule
masses and are genetically identical.
This analysis is performed by digestion of
plasmid with restrictive endonucleases. Techniques
used in plasmid analysis could be direct or
indirect. The direct techniques include (i) DNA
chains hybridization with calculation of homology
degree (ii) direct electronomicroscopic heteroduplex
analysis of small plasmids (nonhomological
DNA would appear to be a simple loop), (iii)
discovering of DNA sequences by Southern-blot
hybridization with radioactive DNA probes. The
indirect techniques are (i) agarose gel electrophoresis
and (i) restriction endonuclease analysis (3).
Comparison of strains, based on their
contents of plasmids or on restrictive profiles of
plasmid DNA, is very useful in the case that a
bacterium contains plasmids. However, when a
bacterium does not contain plasmids, this
method gives us very little benefit.
Plasmid profile analysis is useful in determining
the epidemical strain in outbreaks caused
by multiple species: Escherichia, Klebsiella, Pseudomonas,
Serratia, Staphylococcus and so on
(4). This method allowed successful identification
of an epidemical strain P.aeruginosa isolated
from patient on hemodialysis. Actually, the same
strain was isolated both from patient flora and
from iodoform solution which was associated with
infection spread and removed from market
afterwards (6).
Applying of plasmid profile analysis did
confirm that usual humane infection was
provoked by consumption of animal originated
food. Identical resistance plasmids from Salmonella
serovars isolated from humans and animals,
in the case of Salmonella enterica serovar
Typhimurium (S.Typhimurium) var Copenhagen,
S. Newport, and S. Dublin have been obtained
from different geographic areas in the US.
Thanks to the plasmid profile analysis of these
strains, it has been confirmed that they spread
out by means of using animal-originated food.
The method of restriction endonuclease analysis
has shown that plasmids from animal and human
isolates were quite often identical or almost
identical (7).
In examination of epidemical strains
S.Typhimurium there is a common opinion that
method of plasmid profile analysis is easy to use,
equally specific as phagotypization, and superior
against biotypization and resistotypization (8).
Plasmid profile analysis has enabled discovering
of infection source associated with multiresistant
(MR) strains of S.Typhimurim in Sao Paolo
(Brazil). It is established that infections associated
with strains with the same plasmid profile
occurred among children hospitalized in the same
hospital, thus endemic process consisted of
series of small outbreaks raised by infection
among children in some hospitals.
Plasmid profile analysis has proved as
successful method in solving of outbreaks caused
by S.Typhimurium in our laboratories, too. In an
outbreak associated with this serovar occurred in
a military base, Cobeljic et al. have detected
epidemic strain in plasmids of 30 MDa and 60
MDa. The strain was isolated from the patient’s
stool, from the cook as a carrier of infection and
from food. At the same time, analyzing the other
outbreak in the base, the same authors pointed
out that strains of the same serovar could be
isolated from the outbreak, which were not
associated with it (10).
Plasmid profile analysis enables solving
outbreaks caused by other salmonellas. In an
outbreak associated with S.Newport, in the US, it
was established that a beef from the farm was
the origin of epidemic since each strain, isolated
from patients had R plasmid which conditioned
resistance to ampicillin, carbencillin, and tetracycline.
Though many people were infected, only
those, who did allow selective advantage and
growth by antibiotics, became ill (11).
In several US states, plasmid profile in
strains isolated from patient’s stools with
enterocolitis caused by S. Muenchen, and from
marihuana, allowed identification of an epidemic
strain with unusual way of spreading (12). At the
Health Care Institute in Novi Sad, there were
isolates of S. Hadar, associated with an outbreak
and obtained from patient’s and the restaurant
employees stools, and from food, resistant to
ampicillin and streptomycin. Plasmid profile
analysis revealed presence of five plasmid stripes
with molecule masse of 13, 5.4, 4.2, 2.0, and 1.7
MDa (13).
By plasmid profile analysis, we can
determine a different number of profiles, which
depends on geographic location of isolates, time
of researching, as well as the origin of strains,
that is, whether they are isolated from human,
animal or from food. In 2005, in Scotland, in 523
of 543 isolates (96.3%) of S. Enteritidis, which is
one of the most common serovar, there was
found one plasmid at least. In 269 of these
isolates (49.5%), there was only virulence
plasmid of 57 kb, and twenty isolates did not
contain any plasmid. At 134 PT1 isolates there
were described 16 different plasmid profiles, and
11 profiles were found in 117 PT4 isolates. In the
rest of 292 isolates of S. Enteritidis, there were
found 74 different plasmid profiles (14).
Investigating strains of S. Enteritidis
isolated from people and different animals, there
were identified sixteen different plasmid profiles
which were used to make a difference between
strains especially among most common
phagotypes (PT 8 and PT 13a) (15). In the study
carried out on 318 strains of S. Enteritidis
isolated from poultry and its environment in
Canada, there were evidenced 12 phagotypes.
However, among these strains it was possible to
determine fifteen different plasmid profiles (16).
Investigating plasmid profile of S.
Enteritidis isolated from poultry during 1989 –
‘90 in Canada, the authors have proved twelve
plasmid profiles against just four phagotypes
55

Plasmid profile analysis of salmonella enterica serotype enteritidis Biljana Miljković-Selimović et al.
(17). Investigating 105 strains of S.Enteritidis
(72 of them had human origin and 33 non-
human origin) isolated in the period from 1975 to
1995, seven different plasmid profiles were
proved, while 96% isolates had have plasmid of
around 36 MDa (18). Investigating 64 isolates of
S.Enteritidis from laboratory collection of Enterobacteriaceae
at the University of Medical Science
in Ankara, 88% of examined isolates had from 1
up to 4 plasmids which sizes varied from 2.5 to
100 kbp. Plasmid of 57 kbp was the most common
and it was proved in 69% of isolates as a single one
or along with the other plasmids (19).
Plasmid profile is determined for 276
strains of S.Enteritidis isolated at the Public
Health Institute in Nis. From 94 isolates there
were investigated 28 epidemic and 182 non-
epidemic strains. Plasmid profile analysis of
epidemic and non-epidemic strains showed up to
12 plasmid profiles. Among the two most
common plasmid profile: PP 38 MDa and PP 38
2MDa, plasmid profiles with plasmid sizes of 4.0,
3.4, 2.7, and 1.5 MDa were determined too, as
well as PP with large plasmids of 34 and 27Mda,
though scattered (20).
Plasmid profile analysis was successfully
applied to resolving outbreaks associated with
S.Enteritidis. Researching the outbreak of
enterocolitis at restaurant chain in Meriland,
plasmid profile (55, 3.5, and 2.5 kb) was
determined in isolates of S.Enteritidis isolated
from every patient. The probability that this
plasmid profile was randomly associated to the
outbreak was less than 0.05 (21). By researching
of an outbreak at the University of Medical
Science in Ankara, it was determined that
outbreak strains carried the same plasmid
profile: three plasmids with size of 57, 40, and
3.0 kbp (19). An outbreak caused by S.Enteritidis
phagotype 13 (S. Enteritidis PT13) occurred in
Czech Republic. The strain belonged to rare
phagotype and was susceptible to ampicillin. Both
pre-epidemic and post-epidemic isolated strains
were inspected by method of plasmid profile
analysis. It was determined that ampicillin
resistance was associated with plasmid of 200
kb, which was evidenced at S. Enteritidis (20).
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clone concept in the epidemiology, taxonomy, and
evolution of the Enterobacteriaceae and other
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2. Riley LW, DiFerdinando GT Jr, DeMelfi TM, Cohen
ML. Evaluation of isolated cases of salmonellosis by
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3. Farrar WE Jr. Molecular analysis of plasmids in
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56
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of PT 8 strains because it was possible to
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ANALIZA PLAZMIDSKOG PROFILA SALMONELLAE ENTERICA SEROTIP
ENTERITIDIS
Biljana Miljković-Selimović, Tatjana Babić, Branislava Kocić, Predrag Stojanović, Ljiljana Ristić i Marina
Dinić
Analiza plazmidskog profila (PP) je određivanje broja i veličine plazmida u bakterijskim
izolatima. Prilikom primene analize plazmidskog profila u određivanju epidemijskog soja za
procenu ove metode, neophodno je porediti epidemijske sojeve sa neepidemijskim
sojevima izolovanih u istom vremenskom periodu. Određivanje plazmidskog profila vrši se
lizom bakterijske ćelije, denaturacijom hromozoma, taloženjem ćelijskih ostataka
centrifugiranjem i precipitacijom DNK etanolom. DNK se razdvaja elektroforezom u gelu na
osnovu molekulske mase. Plazmidi mogu biti analizirani i restrikcionim enzimima, ali i
direktnim tehnikama, hibridizacijom i elektronomikroskopski. Primena analize plazmidskog
profila je omogućila rešavanje brojnih epidemija izazvanih crevnim bakterijama, naročito
salmonelama. Kada je u pitanju Salmonella enterica serotip Enterititidis, metoda se može
koristiti sama, kao metoda komplementarna fagotipizaciji, ali se može dopunjavati i
drugim molekularno-genetskim tehnikama. Acta Medica Medianae 2008;47(2):54-57.
Ključne reči: plazmidski profil, Salmonella enterica serotip Enteritidis
57